Our long range goal is to understand, at the molecular level, how transcription Of eukaryotic protein-coding genes is initiated and regulated. The goal of the research described in this proposal is to determine the enzymatic mechanism of transcription catalyzed by mammalian RNA polymerase II and a set of general transcription factors that direct accurate initiation and efficient elongation of transcripts synthesized from a large number of promoters. We have focused on rat liver as a model for our studies of the enzymology of transcription by RNA polymerase II because the general factors can be purified to homogeneity from rat liver in quantities sufficient for serious biochemical analyses of their structures and mechanisms of action. The general aims guiding the research described in this proposal remain essentially the same as those in the original proposal: (i) to reconstitute in vitro, with purified enzymes, faithful transcription of eukaryotic protein-coding genes and (ii) to use this reconstituted enzyme system to elucidate the biochemical mechanisms governing transcription initiation and elongation by mammalian RNA polymerase II. The research will be organized along the following lines: (1) Detailed analysis of the structure and function of a novel RNA polymerase II elongation factor designated Elongin (SIll) and its interactions with the product of the von Hippel-Lindau (VHL) tumor suppressor gene. (2) Purification and analysis of a newly identified elongation factor designated 5-IV. (3) Detailed analysis of the role of TFIIF in selective binding of RNA polymerase II to its promoters. (4) Investigation of the role of TFIIH in ATP-dependent activation of transcription initiation. These studies should provide a solid foundation for future investigations of the mechanisms governing eukaryotic messenger RNA synthesis, and they should also yield significant insights into the molecular basis of von Hippel-Lindau disease and its associated cancers.